Diagnostic reagents for human cytomegalovirus and methods of use

ABSTRACT

The invention provides a plurality of peptides (and immunologically functional variants thereof) which are immunogenic epitopes recognized by CD8 +  class I MHC restricted cytotoxic T-lymphocytes of patients harboring latent human cytomegalovirus (HCMV) infection. The peptides are capable of activating CTLs and CTLps in the absence of active viral replication, and thus are useful for eliciting a cellular immune response against HCMV by normal and immunodeficient subjects. Peptide and lipopeptide vaccines, with and without adjuvants, also are disclosed. Cellular vaccines comprising the peptides form a further embodiment of this invention.

[0001] This application is a continuation of prior copending applicationSer. No. 09/692,170, filed Oct. 20, 2000, which is acontinuation-in-part of prior copending application Ser. No. 09/534,639,filed Mar. 27, 2000 (U.S. Pat. No. 6,251,399), which is a divisional ofSer. No. 09/075,257, filed May 11, 1998 (U.S. Pat. No. 6,074,645), whichis a continuation-in-part of co-pending application Ser. No. 09/021,298,filed Feb. 10, 1998 (U.S. Pat. No. 6,156,317), which is acontinuation-in-part of application Ser. No. 08/950,064, filed Oct. 14,1997, now abandoned, which is a continuation-in-part of application Ser.No. 08/747,488, filed Nov. 12, 1996, now abandoned.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support in the form ofgrant nos. CA30206, CA77544 and CA33572 from the United StatesDepartment of Health and Human Services, National Cancer Institute. Thegovernment may have certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] 1. Technical Field

[0004] This invention relates to human cytomegalovirus (HCMV), and inparticular to peptide fragments from one or more subunit proteins thatfunction as T-cell epitopes of HCMV in human beings. The peptides ofthis invention are capable of directing human cytotoxic T lymphocytes(CTL) to recognize and lyse human cells equivalently to infection withHCMV. Vaccines formulated using those peptides also are provided by thisinvention.

[0005] 2. Description of the Background Art

[0006] The HCMV genome is relatively large (about 235 k base pairs) andhas the capacity to encode more than two hundred proteins. HCMV iscomposed of a nuclear complex of double-stranded DNA surrounded bycapsid proteins having structural or enzymatic functions, and anexternal glycopeptide- and glycolipid-containing membrane envelope. HCMVis a member of the herpes virus family and has been associated with anumber of clinical syndromes.

[0007] HCMV infection is relatively common and is usually self-limitingin the healthy, immunocompetent child or adult (L. Rasmussen, Curr. Top.Microbiol. Immunol. 154:221-254, 1990), however, approximately 10% ofall newborn infants carry HCMV and the virus can cause severe congenitaldisease in the fetus or infant. Some of these newborn infants suffercongenital birth defects. Other newborn infants carry cytomegalovirusfor some time before they show symptoms of disease. HCMV is a commoncause of mental retardation in children who acquire the infection inutero from mothers carrying an active infection.

[0008] Several studies have begun to question whether persistent andapparently asymptomatic HCMV infection in an otherwise healthy adultposes health risks in certain individuals. For example, individuals whohave undergone coronary angioplasty sometimes subsequently developrestenosis as a result of arterial remodeling. In one study, about onethird of such patients with restenosis had detectable HCMV DNA in theirarterial lesions (E. Speir et al., Science 265:391-394 (1994)). Inanother study, CMV seropositive patients were five times more likely todevelop restenosis than their seronegative counterparts (Y. F. Zhou etal., New England J. Med. 335:624-630 (1996)). These studies suggest thatdecreasing the number of HCMV infected host cells can benefit certainindividuals.

[0009] HCMV also has been associated with morbidity and mortality inimmunocompromised patients. HCMV is an important consideration in thetreatment of patients suffering from Acquired Immunodeficiency Syndrome(AIDS). The defining complication is viral retinitis, which, if leftuntreated, can lead to blindness. Historically, CMV disease has been oneof the more devastating of the opportunistic infections that besetHIV-1-infected individuals. Disease manifestations of CMV viremia whichappear as the CD4⁺ T cell counts drops below 100/mm³ includeencephalitis, enteritis and pneumonia. At autopsy there is multi-organinvolvement of CMV disease in the preponderance of AIDS patients who hadsevere CMV retinitis. Patients infected with HCMV often sufferimpairment of some of their vital organs, including the salivary glands,brain, kidney, liver and lungs. Furthermore, HCMV is associated with awide spectrum of classical syndromes including mononucleosis andinterstitial pneumonia. HCMV also has an oncogenic potential and apossible association with certain types of malignancies includingKaposi's sarcoma.

[0010] HCMV can cause opportunistic infections resulting in a variety ofcomplications in, for example, immunosuppressed organ transplantpatients. Prior to the use of antiviral chemotherapy, HCMV infection hadbeen responsible for a substantial proportion of post-bone marrowtransplantation (BMT) complications (J. Meyers et al., J. Infect Dis.153:478-488 (1986)). The advent of drugs such as ganciclovir withsubstantial anti-HCMV activity dramatically reduced complicationsassociated with post-BMT CMV infections (G. Schmidt et al. New EnglandJ. Med. 324:1005-1011 (1991); J. M. Goodrich et al., New England J. Med.325:1601-1607 (1991)).

[0011] Ganciclovir is most effective when administered prophylacticallybefore diagnosis of HCMV infection. This approach has several negativeconsequences, however, including a higher proportion of recipientsbecoming neutropenic (one third) and increased numbers of concomitantfatal bacterial and fungal diseases (J. M. Goodrich et al., Ann. Intern.Med. 118:173-178 (1993)). An alternative approach in which ganciclovirwas given when HCMV antigens or DNA are first detected by culturemethods provided no survival advantage compared to prophylaxis ortreatment post-disease for all patients (D. J. Winston et al., Ann.Intern. Med. 118:179-184 (1993)). Finally, because of the acute natureof the side-effects, there is a need for increased hospitalization andgrowth factor administration to treated patients which, coupled with thecost of ganciclovir prophylaxis, increases the cost of BMT after-care.

[0012] Because human cytomegalovirus is relatively common, yet isassociated with extremely serious health conditions, a considerableeffort has been made to study the biology of the virus with the aims ofimproving diagnosis of the disease as well as developing preventativeand therapeutic strategies. The mounting of a CD8⁺ CTL response isbelieved to be an important mammalian host response to certain acuteviral infections. The observations that HCMV infection is widespread andpersistent, and can become reactivated and clinically evident in theimmunosuppressed patient, suggest that virus-specific T-cells, includingHCMV-specific CTL, play an important role in the control of persistentinfection and in recovery from HCMV disease.

[0013] In humans, protection from the development of CMV disease inimmunosuppressed BMT recipients correlates with the recovery ofmeasurable CD8⁺ CMV-specific class I MHC-restricted T cell responses(Quinnan et al., New Eng. J. Med. 307:7-13 (1982); Reusser et al., Blood78:1373-1380 (1991)). These observations led investigators to carry outclinical trials in which donor-derived HCMV-specific CD8⁺ CTL wereinfused into BMT recipients as an alternative to ganciclovir prophylaxisand therapy (S. R. Riddell et al., Science 257:238-241 (1992)). Thetransfer of CD8⁺ CTL clones to allogeneic bone marrow transplantrecipients resulted in detectable CTL-based HCMV immunity, andstatistically significant diminution of HCMV disease after BMT (E. A.Walter et al., New Eng. J. Med. 333:1038-1044 (1995)).

[0014] Although successful in application, this approach has thedisadvantage that it requires a sophisticated laboratory setup (which isalso highly labor-intensive and costly) to derive the HCMV-specific CTLin vitro for reinfusion into a patient. A desirable alternative would beto deliver a vaccine derived from HCMV that would impart immunity to aBMT recipient, a solid organ recipient, a heart patient, an AIDS patientor a woman of child-bearing years, without the need for ex vivoexpansion of HCMV-specific CTL. No such vaccine presently is available,however. To develop such a vaccine, the viral peptide which cause thehost to recognize HCMV in a protective manner must be identified, sothat their amino acid sequence information can be determined.

[0015] The viral life cycle provides insight as to the most effectivetime frame for targeting a vaccine to maximally disrupt virus productionand spread. Following HCMV entry into the host cell and uncoating, theviral genome is expressed sequentially via immediate early (0-2 hour),early (2-24 hour) and late (>24 hour) viral proteins. However, certainviral structural proteins such as pp65 are chaperoned into the cellbecause of their existence in large quantity in the viral particle. Muchattention has focused upon structural virion proteins as potentialimmunodominant target antigens for HCMV-specific CTL responses.

[0016] One viral structural protein, pp65, has been identified as atarget antigen for CMV-specific class I MHC restricted CTL derived fromthe peripheral blood of most asymptomatic CMV seropositive individuals(E. Mclaughlin-Taylor et al., J. Med. Virol. 43:103-110 (1994)). TheImmediate Early (IE) protein is processed to form CTL epitopes whichcause the stimulation of IE-specific CTL in many normal blood donors (F.Kern et al., J. Virol. 73(10):8179-8184 (1999); C. Retiere et al., J.Virol. 74(9):3948-3952 (2000); G. Zyulai et al., J. Infect. Dis.181:1537-1546 (2000); F. Kern et al., Eur. J. Immunol. 30:1676-1682(2000). Nevertheless, it has not been shown that IE-specific CTL kill orlyse CMV-infected target cells similarly to CTL specific to structuralproteins such as pp65 or pp150. Importantly, CD8⁺ class I MHC restrictedCTL specific for pp65 will recognize autologous HCMV-infected cellswithout the requirement for viral gene expression, presumably as aresult of processing of the internal depot of pp65 that is transferredinto the cell during infection (M. J. Gilbert et al., J. Virology67:3461-3469 (1993)). CTL against pp65 or pp150(another matrix proteinthat is recognized frequently) are able to recognize and lyseHCMV-infected cells in vitro within an hour of infection in the absenceof viral gene expression (S. R. Riddell and P. D. Greenberg, Curr. Top.Microbiol. Immunol. 189:9-34 (1994)). Thus, these CTL may represent animportant effector cell for limiting HCMV reactivation and progressionto CMV disease, and such a cellular immune response in bothimmunocompromised and normal individuals would be extremely important(C. -R. Li et al., Blood 83:1971-1979 (1994)). CTL recognizing envelopeproteins are not a substitute for pp65 and pp150 CTL because they arerarely found, arising late in infection and they are poor lyticeffectors because of the down-regulation of the required Class I MHCmolecules (M. J. Gilbert et al., J. Virology 67:3461-3469 (1993)).Therefore, vaccines stimulating immunity against pp65 or pp150 may bethe preferred mechanism for eliciting protective immunity against CMVinfection.

[0017] Individual MHC Class I molecules preferentially bind peptides ofa given motif. The amino acid sequence of specific positions of themotif are invariant, allowing a given peptide to bind to MHC Class Imolecules with high affinity. These are referred to as “anchorpositions” (K. Falk et al., Nature 351:290-296 (1991)). Amino acidpositions other than the anchor positions also contribute to thespecificity of peptide binding to MHC Class I molecules. Additionally,residues at positions within the CTL epitope which do not interact withMHC may nevertheless interact with T cells, presumably by binding the TCell receptor (TCR). The binding of peptide amino acid residues to MHCor TCR structures is independently governed, so that substitution of TCRbinding amino acid residues in many cases will not interfere withbinding to the MHC molecule on the surface of an antigen presentingcell.

[0018] Edman degradation followed by N-terminal sequence analysis hasbeen used to sequence the peptide mixture which is bound to the MHCclass I peptide binding groove. In most cases the length of thesepeptides is between 9 and 11 amino acids. Mass spectrometry of HPLCseparated peptide mixtures can elucidate the primary sequence ofindividual peptides. Peptide fragments which bind to MHC identified inthis manner are referred to as “naturally processed epitopes.”Alternatively, one can predict which peptides of a given length, between9-11 amino acids, will optimally bind to individual HLA Class I allelesbased on their conformity to a motif (K. Falk et al., Nature 351:290-296(1991)). One such motif has been established for HLA A*0201. Nonapeptideanchor residues are located at positions 2 and 9 for HLA A*0201, withminor contributions to binding from positions 1, 4, 3, 5, 6, 7, 8 indecreasing order of importance to binding strength (J. W. Drijfhout etal., Human Immunology 43:1-12 (1995)). Similar motifs have beenestablished for decamers and undecamers for HLA A*0201. Correspondingly,unique amino acid motifs have been established for a subset of other HLAA and B alleles to predict binding peptides between 8-11 amino acids (H.G. Rammensee et al., Immunogenetics 41 (4):178-228 (1995)).

[0019] It is recognized that CTL are an important mechanism by which amammalian organism defends itself against infection by viruses andpossibly cancer. A processed form of, e.g., a viral protein minimalcytotoxic epitope (MCE) in combination with MHC Class I molecules isrecognized by T cells, such as CD8⁺ CTL. Functional studies of viral andtumor-specific T cells have confirmed that an MCE of 8-12 amino acidscan prime an antigen presenting cell (APC) to be lysed by CD8⁺ CTL, aslong as the APC expresses on the cell surface the correct MHC moleculethat will bind the peptide.

[0020] It has been shown that the route of entry of a protein into thecell determines whether it will be processed as an antigen bound toeither MHC Class I or II molecules. The endogenous or Class I pathway ofprotein degradation is often used by infectious viruses when they arepresent within cells. Viral nucleoproteins which may never reach thecell surface as full length molecules are still processed within thecell, and degraded portions are transported to the surface via MHC ClassI molecules. Viral envelope glycoproteins, merely because they are cellsurface molecules, do not obligatorily induce CTL recognition. Rather,viral nucleoproteins, predominantly in the form of processed epitopes,are the target antigens recognized by CD8⁺ CTL (A. Townsend et al.,Philos. Trans. R. Soc. Lond. (Biol). 323:527-533 (1989)).

[0021] Antigens entering the cell through exogenous pathways(pinocytosis, etc.) are not typically processed and presented by Class IMHC molecules. Therefore, methods to introduce proteins directly intothe cytoplasm have become a focus of vaccine developers. An approachthat has gained favor is to use recombinant vaccinia viruses to infectcells, delivering a large amount of intracellular antigen. Theenthusiasm for using vaccinia viruses as vaccines has diminished,however, because these viruses have the potential to cause disease inimmunosuppressed people, such as BMT recipients based on the WesternReserve strain. Another approach to vaccination is to mix an antigenicprotein with an adjuvant and introduce the mixture under the skin bysubcutaneous injection.

[0022] Yet another potential approach to immunization to elicit CTL isto use the MCE defined for a viral antigen in the context of aparticular MHC restriction element to boost a CTL memory response to avirus. The ability of an MCE to provide protective immunity to challengeby a lethal dose of an infectious virus has been discussed in theliterature. Vaccine developers have developed increasing interest inutilizing the MCE as the vaccine because it is capable of binding to MHCClass I molecules through external binding of the cell surface moleculeswithout internalization or processing.

[0023] Historically, the MCE has been most effective as an immunogenwhen synthesized as a lipidated peptide together with a helper CD4epitope (A. Vitiello et al., J. Clin. Invest. 95:341-349 (1995) and B.Livingston et al., J. Immunol. 159:1383-1392, 1997). Other modificationsof the bispecific vaccine include inclusion of a signal sequence forendoplasmic reticulum retention and targeting (KDEL) to attain maximumactivity. There is also evidence in the literature that an MCE presentedby particular types of APC (e.g. dendritic cells) may cause a primaryimmune response to occur in the absence of viral infection or priorcontact with the virus or tumor cell.

[0024] Introduction of CMV-specific CTL into a recipient is not auniversally applicable and practical strategy to confer immunity to allthose at-risk individuals who may need to be immunized against HCMVinfection. Accordingly, in spite of significant efforts towardsidentifying the HCMV proteins that are recognized by CTLs, as well asthe specific identification of the HCMV late structural protein pp65,improved methods of preventing and treating HCMV infection are needed.

SUMMARY OF THE INVENTION

[0025] Accordingly, one aspect of the present invention relates toimmunologically active peptides, and functional variants thereof,capable of eliciting a cellular immune response to HCMV in humans, e.g.SEQ ID NOS: 10-25 and 30-41. The peptides are capable of directing humanCTL to recognize and lyse human cells infected with HCMV. Suchimmunologically active peptides, in association with an MHC Class Imolecule, are recognized by CTL of individuals having a latent(inactive) HCMV infection.

[0026] Another aspect of the present invention provides a method ofaugmenting the immune system of a patient in need thereof (i.e., apatient harboring a latent or active CMV infection) by administering atleast one immunologically active peptide or peptide derivative accordingto the present invention that will be recognized by CTL and/or CTLp (CTLprecursors) of the patient.

[0027] In yet another aspect of the invention, at least oneimmunologically active peptide is administered to uninfected individualsto provide immunity against future infections by HCMV. Such a peptidemay be administered in the form of a peptide or lipopeptide vaccine,optionally with an adjuvant.

[0028] Alternatively, the peptide(s) may be administered in the form ofa cellular vaccine via the administration of autologous or allogeneicantigen presenting cells or dendritic cells that have been treated invitro so as to present peptide on their surface.

[0029] Yet another aspect of the invention is a method to augment theimmune response of an individual who is latently infected with CMV andis at risk for reactivation of CMV infection, wherein T cells areremoved from an individual and treated in vitro with a peptide of thepresent invention. The resulting CMV-reactive CTL are reinfusedautologously or allogeneically to the patient, for example, a BMTrecipient.

[0030] In yet another aspect, a method to confer immunity against anHCMV infection to a previously uninfected individual includes the stepsof removing T cells from the individual, exposing the T cells in vitroto a peptide of the present invention and then reinfusing the resultingHCMV-reactive CTL to the individual.

[0031] The peptides of the present invention also may be administered topreviously infected or uninfected patients, or in vitro to T cells, inthe form of a polynucleotide (DNA-based) vaccine, wherein a suitablegene transfer vector, such as a plasmid or an engineered viral vectorthat contains DNA encoding the peptide fragment under the control ofappropriate expression regulatory sequences, is administered to thepatient or to T cells in culture.

[0032] In yet another of its aspects, the present invention provides avaccinia, modified Vaccinia Ankara, canarypox or other eukaryotic virusvector containing a DNA sequence encoding the immunologically activepeptide fragment, optionally including a helper CD4 epitope in therecombinant virus to stimulate and prolong CTL function. The vectorinfects an antigen presenting cell which in turn presents antigen thatwill be recognized by CTLs of patients having a latent (inactive) HCMVinfection.

[0033] An additional aspect of the invention relates to diagnosticreagents for detection of the presence of active versus quiescent HCMVinfections. The peptides according to the present invention can directlystimulate CTLp in vitro and therefore can be used in an assay todetermine the degree of immunostimulation being caused by HCMV. Thepeptides can also be used to distinguish individuals who areseropositive from those who are seronegative. T cells from a patient canbe contacted in vitro with antigen presenting cells that have beenprimed with a peptide according to the present invention.

[0034] The invention includes peptides according to SEQ ID NOS: 10-25and 30-41 and vaccines comprising the peptides of SEQ ID NOS: 1, 10-25and 30-41. Cellular vaccines also are provided, which comprise antigenpresenting cells that have been treated in vitro so as to present apeptide according to any one of SEQ ID NOS: 1, 10-25 and 30-41. Further,the invention comprises a recombinant viral vector vaccine whichexpresses a gene encoding a peptide according to any one of SEQ ID NOS:1-14, 19, 26-28 and 30. In addition, a further embodiment comprises amethod of modulating the immune response to human cytomegalovirusinfection, comprising administering a vaccine as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 shows the cytotoxic response elicited by peptides in theabsence of lipidation.

[0036]FIG. 2 shows the cytotoxic response elicited by monolipidatedpeptides.

[0037]FIG. 3 shows the cytotoxic response elicited by dilipidatedpeptides.

[0038]FIG. 4 is bar graphs showing cytotoxicity of antigen presentingcells by immunized splenocytes after one (A), two (B) or four (C) invitro stimulations.

[0039]FIG. 5 presents the results of chromium release assays for cellsimmunized with SEQ ID NO: 37.

[0040]FIG. 6 presents the results of chromium release assays for cellsimmunized with SEQ ID NO: 37.

[0041]FIG. 7 shows comparative cytotoxicity data for cells from miceimmunized with 100 or 25 nmoles SEQ ID NO: 37.

[0042]FIG. 8 compares the cytoxicity data for cells immunized with SEQID NO: 37 in different vaccine formulations.

[0043]FIG. 9 compares the effectiveness of vaccines containing SEQ IDNO: 37 and differing concentrations of DMSO.

[0044]FIG. 10 compares cytotoxicity data from cells immunized withdifferent vaccine formulations of SEQ ID NO: 37.

[0045]FIG. 11 compares cytotoxicity data from cells immunized with 25 or50 nmoles SEQ ID NO: 37 with or without a booster immunization.

[0046]FIG. 12 compares cytotoxicity data from cells immunized with SEQID NO: 37 in different formulations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] A nonapeptide having the sequence NLVPMVATV (pp65₄₉₅₋₅₀₃) (SEQ IDNO: 1) is an immunogenic epitope of pp65 from CMV laboratory strainsAD169 and Towne and all wild type isolates examined to date. The epitopeis recognized by CD8⁺ Class I MHC restricted cytotoxic T-lymphocytes ofpatients harboring latent CMV infection. The peptide is capable ofactivating CTL in the absence of active viral replication, and thus isuseful for augmenting the immune system of both normal andimmunodeficient patients, as well as in the study of the Class I antigenprocessing pathway for HCMV proteins. The amino acid residues inpositions 2 and 9 are the preferred residues at those positions forinteraction with HLA A*0201 and certain subtypes of HLA A*02XX, whereXX=subtypes 02-22 (J. W. Drijfhout et al., Human Immunology 43:1-12(1995)). Nonetheless, other less preferred amino acid residues mayreplace the preferred anchors, and the peptide can continue to exhibitthe capacity to bind HLA A*0201 and certain subtypes of HLA A*02XX andto stimulate HCMV-specific CD8⁺ CTL.

[0048] Thus, in one aspect, the present invention provides animmunologically active peptide, capable of eliciting a cellular immuneresponse to human cytomegalovirus infection, of the preferred sequence:

[0049] NLVPMVATV (SEQ ID NO: 1).

[0050] Sequence variants of the preferred peptide include peptides ofthe sequence NX₁VPMVATX₂ wherein X₁ is L,I,M,T or V, and X₂ is V,A,C,I,Lor T (SEQ ID NO: 2). The invention includes the construction andselection of other functional sequence variants, which can be carriedout by those skilled in the art based upon the present disclosure. Thepeptide or the structural variants disclosed herein also can be afunctional part of a longer peptide which produces the immunologicaleffects disclosed herein, for example AAKXVAAWTLKAAANLVPMVATV wherein Xis cyclohexylalanine; SEQ ID NO: 15 or (d)AAKXVAAWTLKAAANLVPMVATV whereX signifies cylohexylalanine (SEQ ID NO: 16).

[0051] Other immunologically active peptides according to the presentinvention include the peptides:

[0052] YSEHPTFTSQY (SEQ ID NO: 3)

[0053] which binds to HLA A*01XX including A*0101 and subtypes thereof.Sequence variants of this peptide include peptides of the sequenceYXEHPTFTSQY wherein X is S, T or L (SEQ ID NO: 4). The inventionincludes the construction and selection of other functional sequencevariants, which can be carried out by those skilled in the art basedupon the present disclosure. The peptide or the structural variantsdisclosed herein also can be a functional part of a longer peptide whichproduces the immunological effects disclosed herein.

[0054] FVFPTKDVALR (SEQ ID NO: 5)

[0055] which binds to HLA A*68XX including A*6801 and subtypes thereof.Sequence variants of this peptide include peptides of the sequenceFX₁FPTKDVALX₂ wherein X₁ is V or T and X₂ is L, R or K (SEQ ID NO: 6).The invention includes the construction and selection of otherfunctional sequence variants, which can be carried out by those skilledin the art based upon the present disclosure. The peptide or thestructural variants disclosed herein also can be a functional part of alonger peptide which produces the immunological effects disclosedherein.

[0056] TPRVTGGGAM (SEQ ID NO: 7)

[0057] which binds to HLA B*07XX including B*0702 and subtypes thereof.Sequence variants of this peptide include peptides of the sequenceTPRVTGGGAX wherein X is L, F, or M (SEQ ID NO: 8). The inventionincludes the construction and selection of other functional sequencevariants, which can be carried out by those skilled in the art basedupon the present disclosure. The peptide or the structural variantsdisclosed herein also can be a functional part of a longer peptide whichproduces the immunological effects disclosed herein.

[0058] FPTKDVAL (SEQ ID NO: 9)

[0059] which binds to HLA B*35XX including B*3502, B*3504, B*3506 andother subtypes thereof with compatible peptide binding sites. Theinvention includes the construction and selection of other functionalsequence variants, which can be carried out by those skilled in the artbased upon the present disclosure. The peptide or the structuralvariants disclosed herein also can be a functional part of a longerpeptide which produces the immunological effects disclosed herein.

[0060] RPHERNGFTVL (SEQ ID NO: 10)

[0061] which binds to HLA B*07XX including B*0702 and other subtypesthereof with compatible peptide binding sites. The invention includesthe construction and selection of other functional sequence variants,which can be carried out by those skilled in the art based upon thepresent disclosure. The peptide or the structural variants disclosedherein also can be a functional part of a longer peptide which producesthe immunological effects disclosed herein.

[0062] SVLGPISGHVLK (SEQ ID NO: 11)

[0063] which binds to HLA A*11XX including A*1101 and other subtypesthereof with compatible peptide binding sites. The invention includesthe construction and selection of other functional sequence variants,which can be carried out by those skilled in the art based upon thepresent disclosure. The peptide or the structural variants disclosedherein also can be a functional part of a longer peptide which producesthe immunological effects disclosed herein.

[0064] PTFTSQYRIQGKL (SEQ ID NO: 12)

[0065] which binds to HLA B*3801/2 and other subtypes thereof withcompatible peptide binding sites. The invention includes theconstruction and selection of other functional sequence variants, whichcan be carried out by those skilled in the art based upon the presentdisclosure. The peptide or the structural variants disclosed herein alsocan be a functional part of a longer peptide which produces theimmunological effects disclosed herein.

[0066] EFFWDANDIY (SEQ ID NO: 13)

[0067] which binds to HLA B*44XX including B*4402 and other subtypesthereof with compatible peptide binding sites. The invention includesthe construction and selection of other functional sequence variants,which can be carried out by those skilled in the art based upon thepresent disclosure. The peptide or the structural variants disclosedherein also can be a functional part of a longer peptide which producesthe immunological effects disclosed herein.

[0068] FTSQYRIQGKL (SEQ ID NO: 14)

[0069] which binds to HLA A*24XX including A*2402 and other subtypesthereof with compatible binding sites. The invention includes theconstruction and selection of other functional sequence variants such asTFTSQYRIQGKL (SEQ ID NO: 26), which can be carried out by those skilledin the art based upon the present disclosure. The peptide or structuralvariants disclosed herein also can be a functional part of a longerpeptide which produces the immunological effects disclosed herein.

[0070] The following constructs exemplify peptide vaccines according tothe invention and are not intended to be limiting:AKXVAAWTLKAAANLVPMVATV (SEQ ID NO:17) dAKXVAAWTLKAAANLVPMVATV (SEQ IDNO:18) VSTIVPYIGPALNIAAANLVPMVATV (SEQ ID NO:19)AKXVAAWTLKAAAYLVPMVATV-NH₂ (SEQ ID NO:20) dAKXVAAWTLKAAAYLVPMVATV-NH₂(SEQ ID NO:21) AKXVAAWTLKAAANLVPMVATV-NH₂ (SEQ ID NO:22)AKXVAAWTLKAAAYLVPMVASV-NH₂ (SEQ ID NO:23) AKXVAAWTLKAAANLLPMVASV-NH₂(SEQ ID NO:24) AKXVAAWTLKAAASVLGPISGHVLK (SEQ ID NO:25) wherein X= cyclohexylalanine and d = dextro.

[0071] Truncations of the pp65 (Diamond et al. 1997) protein expressedin vaccinia viruses were screened against pp65-specific T cell (CD8⁺)clones from HLA typed CMV-seropositive individuals (Walter et al., NewEngl. J. Med. 333:1038-1044 (1995); McLaughlin-Taylor et al., J. Med.Virol. 43:103-110 (1994). To facilitate fine scale mapping of CTLepitopes, recombinant vaccinia viruses have been prepared that expresspp65 fragments separated by 100-200 nucleotides throughout the pp65gene, with both amino and carboxyl terminal deletions. When the regioncircumscribed by recombinant vaccinia viruses was no greater than 100amino acids, a series of overlapping peptides were synthesized. A seriesof 15 mer peptides from pp65, overlapping by three amino acids, wereused to identify CTL epitopes. Longmate et al., Immunogenetics (2000).Screening a 100 amino acid stretch of pp65 in this way requires a totalof 29 different peptides. Autologous and HLA mismatched Epstein-Barrvirus transformed lymphocyte cell lines (antigen presenting cells) weresensitized with the screening peptides at a concentration of 50 μM for1-2 hours, washed and chromated. The relevant CTL clone was incubatedwith the lymphocyte cell lines sensitized with peptide, and a standardchromium release assay was performed to determine the sensitivity oflysis. Peptide(s) which successfully sensitized lymphocytes to lysis bythe pp65-specific T cell clones were then further truncated, both at theamino and carboxyl termini and retested. These steps were repeated untilthe minimal cytotoxic epitope that corresponds to the HLA allele of theT cell clones used was defined. Exemplary epitopes of pp65 are shown inTable 6.

[0072] Using the methods outlined above, peptides suitable for vaccineproduction can be identified for T cell clones from individuals having avariety of haplotypes. In this way, a cocktail vaccine useful tovaccinate large multi-ethnic human populations may be created. If avaccine has broad enough reactivity to be usable for at least 90% ofmost ethnic populations, it is suitable to be used for public health. Amulti-epitope peptide vaccine against HCMV would be very beneficial toseveral different at-risk patient groups including transplantrecipients, AIDS patients, and gestational fetuses, not all of whom havebeen HLA-typed.

[0073] Peptides according to the invention may be formulated as vaccinesaccording to any suitable method. Naked peptides or lipidated peptidesmay be formulated in a suitable adjuvant or any other pharmaceuticallyacceptable carrier. Cellular vaccines may be prepared by any knownmethod. HCMV peptides according to the invention may be administeredalone, or together with a helper peptide such as the polyclonal helper Tlymphocyte peptide, PADRE. The CTL epitope and HTL peptide may beadministered together or separately, but it is preferable to administerthem in close time proximity.

[0074] Alternative vaccines include fusions of the helper CD4 peptideepitope with the CTL epitope. The peptides may be fused in either orderand may contain a linker sequence between them if desired. Examples ofhelper CD4 epitopes are the synthetic sequence PADRE (J. Alexander etal., Immunity 1:751-761 (1994)) and tetanus-specific peptides, howeverany helper T lymphocyte peptide may be used. In some cases, these fusionpeptides may not require additional covalent lipid modification oradjuvant when administered by the subcutaneous, intranasal,intraperitoneal or intravenous routes. Alternatively, single strandedDNA containing CpG motifs or any other adjuvant may be co-administeredto provide increased activity of the fusion peptide when simultaneouslyprovided at limiting concentration.

[0075] A preferred alternative is to join the antigenic peptide to PADREsuch that the two sequences form a single peptide chain. PADRE may bepositioned at the N- or C-terminus of the antigenic HCMV peptide, and alinker sequence may be positioned between the two sequences or in frontof the N-terminal sequence, if desired. A preferred structure for thistype of vaccine peptide is AAKXVAAWTLKAAANLVPMVATV wherein X signifiescylohexylalanine (SEQ ID NO: 15) or (d)AAKXVAAWTLKAAANLVPMVATV wherein Xsignifies cylohexylalanine (SEQ ID NO: 16).

[0076] Peptides of the invention may be lipidated or may lack lipids.Unlipidated peptides, whether incorporating a helper peptide sequence ornot, are contemplated by the invention, as are monolipidated,dilipidated or trilipidated peptide vaccines. Suitable lipids which maybe linked to the peptide sequence include, but are not limited topalmitic acid, stearic acid, myristic acid, lauric acid, capric acid,decanoic acid and the like. Lipids may be attached to the peptides atany location and by any convenient method known in the art.

[0077] Adjuvants may form part of the vaccine formulation. Adjuvantssuch as complete or incomplete Freund's adjuvant, aluminum hydroxide orthe like are contemplated, however a preferred adjuvant, particularlyfor use in humans, is a DNA adjuvant. Single-stranded DNA adjuvantscomprising specific sequences including Cytosine- phosphate-Guanosine(CpG) are known in the art and are contemplated for use with thisinvention. DNA adjuvants lacking these CpG sequences also are usefulwith the invention. An exemplary DNA adjuvant may comprise a 20 mer ofnucleotides with 2 CpG motifs, or any DNA oligomer, generally about 20to about 25 nucleotides long. Increased stability of the sequence may beobtained by substituting phosphate groups in the nucleotide backbonewith two groups to create a phosphoro-thioate backbone rather than aphosphoro-diester backbone.

[0078] Evaluation of HCMV peptide-specific human CTL responses may beperformed in HLA-A2.1/k^(b) transgenic mice, a well-accepted animalmodel, as follows. The mice are injected with the vaccine preparation. Abooster may be given 12 days later if desired. About one week after thefinal immunization, splenocytes are prepared from the spleens ofimmunized mice and cultured. Lipopolysaccharide (LPS) blasts for invitro stimulation of the immunized cells may be prepared asepticallyfrom suspensions of unimmunized mouse spleen cells (1.0-1.5×10⁶cells/mL) by addition of 25 μg/mL LPS and 7 μg/mL dextran sulfate. Thesecells are loaded with the same CTL epitope peptide as was used toimmunize the transgenic mice to create antigen-presenting cells. Theseantigen-presenting cells are used to stimulate the cultured splenocytesfrom the immunized transgenic mice in vitro approximately 12-14 daysafter the final immunization. A second in vitro stimulation, or furtherin vitro stimulations, may be performed if desired. A second stimulationis preferred, and may be performed 5-7 days after the first in vitrostimulation. About a week after the last in vitro stimulation, achromium release assay may be performed to test the ability of theimmunized transgenic splenocytes to specifically recognize and killeither mouse or human target cells loaded with the antigenic peptide ofthe vaccine.

EXAMPLE 1

[0079] Derivation of T-Cell Clones

[0080] Methods for deriving T-cell clones from CMV seropositiveindividuals have been described in the literature (see abovereferences). Forty to fifty milliliter samples of whole peripheral bloodwere obtained from CMV seropositive volunteers (detected by standardantibody methods). The white blood cells (WBCs) were separated usingFicoll-HyPaque (DuPont) density gradient centrifugation. The whole bloodwas first centrifuged for 10 minutes at 1400 rpm in a tabletopcentrifuge to reduce the number of red blood cells. The buffy coat wasdiluted to 12 ml with phosphate buffered saline (PBS), and 6 ml werelayered on top of ½ volume of Ficoll-HyPaque. The top layer was removedafter centrifugation at 2000 rpm in a tabletop centrifuge for 15-30minutes. The interface containing the WBC was removed, diluted in PBSand recentrifuged for 8-12 minutes at 1000 rpm, which caused the WBC topellet. The cells were again resuspended in PBS and washed as above oneadditional time. Four to five million WBC/ml were resuspended in T cellmedium (TCM) with human serum obtained from pooled AB+ (blood group) CMVseronegative donors (HAB).

EXAMPLE 2

[0081] Derivation of LCL Antigen-Presenting Cells

[0082] Simultaneously, an autologous antigen presenting cell line wasprepared by Epstein Barr virus immortalization of PBL (see CurrentProtocols in Immunology, Unit 7.22, Wiley-Liss Press (1993)). Derivingthe CTLs and antigen presenting cells from the same individual ensuredHLA matching between the cell lines.

EXAMPLE 3

[0083] In Vitro Stimulation by HCMV

[0084] To initiate the in vitro stimulation of the WBC, a monolayer ofautologous dermal fibroblasts obtained from the same volunteers as theWBC was established by plating the cells in 12-well plates at 10⁵cells/ml/well in DMEM-10% HAB for 24 hours. After 24 hours in culturethe fibroblasts were infected with CMV virions (AD169 or Towne strain)for 2 hours at a multiplicity of infection of between 1 and 5. Themedium and virus were aspirated from the monolayer, and 1 ml of freshDMEM-HAB was added. The monolayer was incubated in the medium for anadditional 4 hours, following which time, the medium was aspirated. Twomilliliters of medium containing 8-10 million WBC were added per wellcontaining CMV infected fibroblasts. The WBC and fibroblasts werecultured in RPMI-1640 (Irvine Scientific) containing 50 U/ml penicillin,50 μg/ml streptomycin, 4 mM L-glutamine, 25 μM 2-mercaptoethanol, 10 mMHEPES and 10% HAB (TCM-HAB). This was termed the first stimulation, andthe cells were co-incubated for seven days. TCM-HAB was replaced if itbecame spent, and the culture was expanded if there was vigorous cellgrowth.

[0085] The WBC were re-stimulated on day 7 by plating onto a freshmonolayer of HCMV-infected autologous fibroblasts prepared as describedabove. In addition, y-irradiated (2500 rad) autologous PBL (5-fold overWBC) were added as feeder cells, and the medium was supplemented withrecombinant IL-2 (10 IU/ml, Chiron-Cetus) on days 2 and 4 of this secondstimulation. Wells which exhibit rapid cell growth require new mediumcontaining IL-2 as the medium became spent.

[0086] After 12-16 days in culture, the cells were harvested and assayedfor recognition of CMV matrix proteins in a chromium release assay(CRA). The CRA was performed using antigen presenting cells as targetcells which are autologous or HLA-mismatched to the T-cell clone. Thecells were prepared by infection with recombinant vaccinia virusescontaining the DNA for HCMV proteins such as pp28 (pp28vac), pp65(pp65vac) and pp150 (pp150vac) or wild-type virus strain WR.

[0087] After overnight infection, the antigen presenting cells wereincubated with chromium-51, washed, and the assay was carried out asdescribed in Current Protocols in Immunology, Wiley-Liss Press, Unit7.17, (1993). In the CRA, the vaccinia-infected target cells were loadedwith chromium-51 and then mixed with cells from the T-cell clone(effector cells). The cells were mixed at a series of effector : targetcell ratios varying from 20:1 to 1:1. After a 4 hour incubation period,the medium in which the cells were incubated was harvested. The releaseof radioactivity into the medium (R_(e)) was quantitated with a gammascintillation counter. The extent to which infected antigen presentingcells exhibit spontaneous lysis and the release of radioactivity (R_(s))in the absence of CTL was established for each virus vector. The maximumamount of radioactivity incorporated into and releasable by the targetcells (R_(max)) was established by lysis of target cells in a detergent(1% Triton X100; Sigma) solution. Percentage cytotoxicity was expressedas:

100×((R_(e))−(R_(s))/((R_(max))−(R_(s)))

[0088] Assays were deemed unacceptable and were repeated unlessspontaneous release (R_(s)) was less than 30%.

[0089] Analysis of the assay was as described, with a positive resultindicated by specific recognition of pp65vac infected autologous APC. Apositive result for pp65 indicated that, in the tested polyclonalpopulation, there were T cells which recognize the pp65 HCMV proteinexpressed by the virus.

EXAMPLE 4

[0090] Procedure for Identification of the CTL Epitope

[0091] WBC stimulated two times by HCMV on dermal fibroblasts werecloned by limiting dilution in 96 well U-bottom plates as follows. Aftertwo HCMV stimulations, the WBC were depleted of CD4⁺ T cells by negativeselection using incubation with paramagnetic beads conjugated toanti-CD4 antibodies. The resulting population was generally between90-95% CD8⁺, a reliable T cell subset marker, and generally 99% CD3⁺, amarker for most peripheral blood T cells as assayed by either flowcytometry or fluorescence microscopy. This final population was platedat a concentration between 0.3-3 cells per well in a final volume of 150μl. Each well also contained γ-irradiated 1.0-3.0×10⁵ allogeneicperipheral blood mononuclear cells (PBMC) in TCM-HAB supplemented with50-100 IU/ml recombinant IL-2 (Chiron-Cetus) and 0.5 μg/ml PHA (Murex).

[0092] After 3 days of culture, the PHA was diluted 2-fold by exchanging75 μl with fresh culture medium supplemented with rIL-2. The wells weresupplemented with fresh rIL-2 every 3-4 days, and medium was replaced asnecessary. The cells were restimulated 12-14 days later with freshallogeneic PBMC as described above, and the plates were carefullyobserved for growth in individual wells. Visible cell growth indicatedthe need to transfer the expanding T cells to larger wells. T cells wererestimulated every two weeks, and were transferred to progressivelylarger wells. At the stage of accumulation of several million cells,some were cryopreserved, and others were subjected to a further CRA. Inthis CRA, the targets were HCMV infected fibroblasts, uninfectedfibroblasts, or autologous LCL infected with wild type vaccinia orvaccinia virus expressing either pp28, pp65 or pp150. HLA mismatchedfibroblasts and LCL were used as controls. One T cell clone amongseveral tested, which was designated 3-3F4, had the characteristics ofbeing both CMV and pp65-specific, and was reactive only to autologoustargets in a specific manner. Other T cell clones with different HLAphenotypes were initially isolated in the same way, except that theinitial peripheral blood sample came from different volunteers.

[0093] The HLA element which restricted the recognition of the T cellclone 3-3F4 to pp65 was identified. A series of LCL were used as targetsthat were singly autologous with each HLA allele of the 3-3F4 cell line.Each target was separately infected with pp65vac, wild type vaccinia, ornot infected at all. The results showed that only the LCL that wereautologous to the HLA A*0201 allele were being recognized and killed bythe 3-3F4 T cell line. It was also established that 3-3F4 is of the CD8⁺T cell subset, characteristic of CTL which recognize Class I restrictedpeptides. Whether the cell line was monoclonal was tested by carryingout PCR repertoire analysis using a series of 26 human Vβ gene segmentprimers. Only one of 26 primers gave a significant signal, the Vβ13.1primer, thereby demonstrating the apparent monoclonality of the 3-3F4 Tcell clone.

[0094] To identify the precise epitope or peptide recognized by the Tcell clone 3-3F4, a series of vaccinia truncations that deleted the pp65protein from the carboxyl towards the amino terminus was used. A CRA wasconducted utilizing autologous and HLA mismatched LCL targets infectedwith vaccinia viruses expressing truncation products of the pp65protein. This experiment localized a region between amino acids 377 and561 that was necessary for killing of targets. Utilizing a small subsetof amino terminal deletions, the region necessary for killing wasfurther localized between amino acids 477 and 561. Utilizing an indirectkilling assay, a monkey kidney cell line was transfected with themolecular HLA allele HLA A*0201 and portions of the pp65 gene tolocalize the region to a fragment containing amino acids 452-561. Thefinal determination of the peptide sequence that corresponds to thesequence bound by HLA A*0201, and is recognized by T cell clone 3-3F4was done by producing 9-10 amino acid peptides on a Synergy (AppliedBiosystems Model 432) peptide synthesis machine. Using publishedinformation, a series of candidate sequences were determined that hadsignificant characteristics of an HLA A*0201 binding sequence, and werelocated in the region between amino acid 452-561. These were: TABLE 1Amino Acid Sequence of Position Peptide Number Motif Score* ILARNLVPMV(SEQ ID NO:26) 491 A*0201 67 ELEGVWQPA (SEQ ID NO:27) 526 A*0201 59RIFAELEGV (SEQ ID NO:28) 522 A*0201 55 NLVPMVATV (SEQ ID NO:1) 495A*0201 63

[0095] Only one of these peptides (referred to as “the 495 peptide” oras “pp65₄₉₅₋₅₀₃”) (SEQ ID NO: 1) proved capable of priming theautologous LCL to be recognized and killed by the CD8⁺ CTL 3-3F4. Other9-10 amino acid peptides from pp65 that also followed the HLA A*0201motif were tested in the CRA. None showed any activity. All peptideswere examined for purity by HPLC on a Vydac C₁₈ column usingacetonitrile/TFA as the moving phase. They were 70-80% pure or greateron average, and were used as dilutions from 0.1% acetic acid solution.

EXAMPLE 5

[0096] Use of the 495 Peptide to Induce Cell Lysis

[0097] Serial dilutions of the 495 peptide showed no change in activitybetween 10 μM and 0.01 μM in priming autologous LCL for killing by Tcell clone 3-3F4 in a CRA. Half-maximal lysis occurred at close to 0.5nM peptide. The peptide-transporter deficient cell line T2 (D. B.Crumpacker et al., J. Immunol. 148:3004-3011 (1992)), which is HLAA*0201 positive, also was used to test the lower limits of sensitivityof the 3-3F4 T cell clone for recognition of the peptide in a CRA. Itwas found that as little as 0.1 nM peptide caused maximal lysis of T2cells, equivalent to the condition with 10 nM peptide. These experimentsdemonstrate that this minimal cytotoxic epitope is a strong binder tothe HLA A*0201 allele.

[0098] The 495 peptide depicted in Table 1 was prepared on a Synergy(Applied Biosystems Model 432) peptide synthesis machine. Dermalfibroblasts were primed with the peptide, then loaded with chromium-51by incubation with 10 pM of the 495 peptide for 2 hours at 37° C., andfor the final hour with chromium-51. The peptide and chromium-51 werewashed out of the medium.

[0099] T cell clone 3-3F4 (CD8⁺ CTL), derived from an HCMV seropositiveindividual (HLA A*0201 positive) was capable of recognizing theHCMV-infected fibroblasts as well as the peptide-primed fibroblasts in aCRA. Uninfected and unprimed fibroblasts were not recognized or killedby the T cell clone. In addition, an HLA-mismatched fibroblast linewithout the HLA A*0201 allele found on the donor fibroblasts was notrecognized or killed by the T cell clone when it was either infected byHCMV or primed with the 495 peptide. Thus, the 495 peptide of thepresent invention can serve as a substitute for the whole HCMV virus,causing normal T cells to recognize and kill fibroblasts as effectivelyor better than if they were infected with HCMV.

EXAMPLE 6

[0100] Generation of TNF-α by T Cell Clones

[0101] TNF-α is a T cell lymphokine that is cytotoxic for many celltypes and may contribute to the in vivo immune response mounted againstan HCMV infection. Cells of the 3-3F4 T cell clone were incubated withautologous fibroblasts pre-incubated with either whole HCMV virions orprimed with the 495 peptide of Table 1. Twenty four hours later,supernatants from the co-incubated cells were applied to an indicatorcell line in a bioassay as described above. The indicator cell line, aWEHI derivative, is sensitive to the cytotoxic action of TNF-A at thepicomolar level.

[0102] Peptide-primed fibroblasts induced the production of as great orgreater levels of TNF-α from T cell clone 3-3F4 as were theHCMV-infected fibroblasts. The peptide of SEQ ID NO: 1 did not induceTNF-α production by either autologous fibroblasts incubated without Tcells or non-HLA-A*0201 expressing fibroblasts incubated with the T cellclone.

EXAMPLE 7

[0103] Peptide SEQ ID NO: 1 can Induce CTL from PBL of HCMV SeropositiveIndividuals

[0104] PBL from HCMV-seropositive individuals were plated onto SEQ IDNO: 1 peptide-primed autologous fibroblasts or incubated with irradiatedautologous PBL sensitized with SEQ ID NO: 1 peptide for seven days. Theonce-stimulated PBL were re-stimulated in a similar manner, either withor without depletion of CD4 T cells. After two weeks, a chromium releaseassay was performed using as targets either peptide-primed,CMV-infected, or untreated autologous fibroblasts. CD8⁺ T cells lysedsignificant percentages of the peptide-primed and HCMV-infectedfibroblasts or EBVLCL primed with SEQ ID NO: 1 , but not untreatedcells. No autologous fibroblast targets were lysed by CD4-depleted PBLfrom freshly drawn blood under the same conditions. Experiments wererepeated with T cells from 15 healthy adult volunteers. In all but onecase, donor possessing a matching HLA allele had T cells which respondedto SEQ ID NO: 1. Similar experiments were conducted with HIV patients,without in vitro stimulation of the T cells. The majority of HIVpositive donors with a matching HLA allele also possessed T cell cloneswhich could recognize autologous EBVLCL primed with SEQ ID NO: 1.

EXAMPLE 8

[0105] Human Cell Lines which Express HLA A2, with Molecular SubtypesOther than A*0201, are Sensitized to Lysis by the 495 Peptide

[0106] Twelve cell lines were subjected to chromium release assays inwhich they were pulsed with the 495 peptide (pp65₄₉₅₋₅₀₃) nonamer,loaded with chromium-51, and incubated with two different HLA A*0201restricted CTL which recognize the 495 peptide and HCMV. The specificcytotoxicity was calculated and is shown in tabular form below for the 1μM and 1 nM concentrations of the 495 peptide. A plus sign (+)represents cytotoxicity greater than 30%, a plus/minus sign (±)represents cytotoxicity between 5% and 30%, and a minus sign (−)represents cytotoxicity less than 15%. TABLE 2 CTL 3-3F4 CTL VB-57 HLAA2 Subtype 1 μM 1 nM 1 μM 1 nM A*0201 + + + + A*0202 + − + − A*0203 + −− − A*0204 + + + + A*0205 + − + + A*0206 + + + + A*0207 + + + + A*0209 +− + − A*0210 + + + + A*0211 + − + +/− A*0217 + − + +

[0107] The data showed that all tested subtypes functionally bound the495 peptide at 1 μM, and the cell lines were lysed by the HCMV and pp65specific CTL. These data also showed that the cell line subtypes shownhere were capable of being sensitized for lysis by the 495 peptide. Thedata also indicate that cells from individuals who carry these subtypescan be sensitized by the peptide for CTL recognition and lysis, albeitat a higher concentration in most cases than what was found for HLAA*0201. Thus, individuals who carry any of these HLA A2 subtypes canrespond to the 495 peptide as a vaccine.

EXAMPLE 9

[0108] Animal Studies Using the 495 Peptide (SEQ ID NO: 1)

[0109] A transgenic mouse model lacking prior HCMV exposure, the HLAA2.1 mouse (E. J. Bernhard et al., J. Exp. Med. 168:1157-1162 (1988)),was employed to test whether the 495 peptide could stimulate CTLslacking prior virus exposure and function as a vaccine. Theseexperiments involved in vitro analysis using mouse and human celltargets, either infected with viruses or primed with the peptide of SEQID NO: 1 or a non-specific control peptide.

[0110] Three mice were immunized subcutaneously at the base of the tailwith 50 μg of the 495 peptide (pp65₄₉₅₋₅₀₃) or peptide p53₁₄₉₋₁₅₇ fromp53 (M. Theobald et al., Proc. Natl. Acad. Sci. U.S.A. 92:11993-11997(1995)) together with 20 μg of the polyclonal helper T lymphocyte (HTL)peptide (PADRE; SEQ ID NO: 29) (J. Alexander et al., Immunity. 1:751-761(1994)) emulsified in IFA (Incomplete Freund's Adjuvant). After twelvedays, spleens were removed from immunized mice, a splenic suspension wasmade, and the effector cells were restimulated for one week usingP53₁₄₉₋₁₅₇ or pp65₄₉₅₋₅₀₃ peptide sensitized syngeneiclipopolysaccharide-treated splenic blast cells (P. A. Wentworth et al.,Eur. J. Immunol. 26:97-101 (1996)). Thereafter, for subsequent in vitrostimulations, the stimulator cells were Jurkat A2.1 cells, prepared bymild acid treatment and subsequent loading of peptides (Z. Yu et al.,Journal of Surgical Research 69:337-343 (1997)). After two in vitrorestimulation cycles, the murine splenic effector population was testedfor recognition of p53₁₄₉₋₁₅₇ or pp65₄₉₅₋₅₀₃ peptide loaded T2 cells.There was substantial recognition of pp65₄₉₅₋₅₀₃ or P53₁₄₉₋₁₅₇ peptidein mice that had been appropriately immunized, without recognition ofthe non-immunizing peptide.

[0111] It was also demonstrated that murine splenic effectors recognizeendogenously processed pp65 in a chromium release assay with human HLAA*0201 EBVLCL targets infected with pp65Vac (D. J. Diamond et al., Blood90 (5):1751-1767 (1997)). Further proof that the pp65₄₉₅₋₅₀₃ peptidecauses recognition of virus-infected cells came from a chromium releaseassay using HCMV-infected human fibroblasts as targets, and murine CTLderived from the pp65₄₉₅₋₅₀₃ peptide stimulation as the effectorpopulation. HLA A*0201 fibroblasts infected with HCMV were capable oflysis by the CTL, whereas both uninfected and mismatched fibroblastswere not recognized (D. J. Diamond et al. Blood 90 (5):1751-1767(1997)). Taken together, these results clearly showed that the spleniceffector population from a primary immunization with pp65₄₉₅₋₅₀₃ arerecognized endogenously processed pp65 and HCMV in an HLA A*0201restricted manner.

[0112] An additional study demonstrated that the 495 peptide induced along-lived immune response in animals. Twelve mice were simultaneouslyimmunized with the 495 peptide (SEQ ID NO: 1)+PADRE (AKXVAAWTLKAAA; SEQID NO: 29)+IFA, and two additional control mice were immunized withPADRE+IFA alone. At two weeks, 6 weeks, 10 weeks and 14 weeks after theimmunization, the spleens from two immunized mice were analyzed forimmunity against the 495 peptide or a control peptide from human p53(p53₁₄₉₋₁₅₇). In addition, at two and six weeks, mice immunized withoutthe 495 peptide also were sacrificed and their spleen cells analyzed forimmune responses against the 495 peptide or the human p53 peptide.Percent specific cytotoxicity (cytotoxicity of 495 peptidetargets—cytotoxicity of p53 targets) was still at a level of 40% after14 weeks, whereas naive animals did not show any specific cytotoxicityabove 5%. This compared well with recent results from immunizing humanvolunteers with the Theradigm™ lipopeptide (B. Livingston et al., J.Immunol. 159:1383-1392 (1997)) in which there was an average maintenanceof 60% of the initial response 38 weeks after the final of fourimmunizations over an eighteen week period.

EXAMPLE 10

[0113] Lipidated Peptides Incorporating pp65₄₉₅₋₅₀₃ (SEQ ID NO: 1) asIFA-Adjuvant Independent Vaccines in Animals

[0114] Although the use of adjuvants to enhance immunogenicity is acommon strategy in animal studies, there are important limitationsconcerning their use in humans. Therefore, one embodiment of the presentinvention involves peptides which incorporate lipid molecules. Thisstrategy has been efficacious in both animal (K. Deres et al., Nature342:561-564 (1989)) and human clinical studies (A. Vitiello et al., J.Clin. Invest. 95:341-349 (1995)) to avoid having to use adjuvants.

[0115] Either one or two palmitic acid moieties were attached to theamino terminus of the 495 peptide, and a series of immunization studieswere conducted in the transgenic HLA A2.1 mouse. The primary structureof the directly lipidated peptides is shown in Table 3. TABLE 3 PrimaryStructure of Peptides Used to Immunize HLA A2.1 Transgenic Mice LipidAdaptor Epitope Sequence Carboxyl SEQ ID Molecule(s) Sequence (SEQ IDNO:1) Terminus NO: 0 -KSS- -NLVPMVATV- OH 30 1 PALMITIC ACID -KSS--NLVPMVATV- OH 31 2 PALMITIC ACID -KSS- -NLVPMVATV- OH 32

[0116] Table 3. Peptides were synthesized on the ABI 432 (AppliedBiosystems), and purified and analyzed as described (D. J. Diamond etal., Blood 90 (5):1751-1767 (1997)). Palmitic acid (Aldrich) wasdissolved in dimethylformamide, and automatically coupled to the aminoterminal lysine. The amino terminal lysine of the monolipidated form ofthe peptide was protected by two protecting groups, Fmoc and Boc. Onlythe Fmoc group is cleaved during synthesis to allow for a single lipidmoiety to be added. For the dilipidated form, the amino terminal lysinewas protected with two Fmoc groups, therefore allowing two lipidmoieties to be added. Peptides also may be lipidated at other locationsby methods well known in the art and are contemplated for use here.

[0117] Separate mice were immunized with unmodified pp65_(495-503,) andeither the monolipidated or dilipidated forms of the peptide togetherwith the PADRE epitope. Because it has been previously shown thatlipidated peptides will stimulate immunity without co-administration ofFreund's adjuvant (H. Schild et al., Eur. J. Immunol. 21:2649-2654(1991)), immunization of mice with and without emulsifying the peptidesin IFA was compared. In a procedure similar to the procedure describedin Example 9 above, twelve days post inoculation the mice weresplenectomized and the spleen cell population was restimulated twice invitro. The splenic effectors were then tested in a chromium releaseassay with peptide-loaded T2 cells, EBVLCL targets infected withpp65Vac, and HCMV-infected fibroblasts (D. J. Diamond et al., Blood 90(5):1751-1767 (1997)). The results demonstrate that the lipidated formof the 495 peptide, without emulsification in adjuvant, induces animmune response capable of recognizing endogenously-processed pp65 andHCMV-infected human cells. The monolipidated form appeared to induce aweaker immune response (compare with and without IFA), whereas theunmodified free peptide had no activity unless emulsified in IFA. (SeeFIGS. 1-3)

[0118] These data illustrate an advantageous immunization/vaccinationprocedure for use in warm blooded animals, including humans. Theprocedure entails the mixture of two peptides in an aqueous solventsystem (the lipidated 495 peptide plus either PADRE or the 830-843 aminoacid tetanus peptide, Table 3). The mixture is administeredsubcutaneously or via another acceptable and efficacious route to eitherHCMV seropositive or seronegative subjects. An additional set of boosterinjections may be employed to enhance the immunity induced by primaryvaccination.

EXAMPLE 11

[0119] Formulation of Lipidated Vaccines

[0120] The 495 peptide and functional sequence variants thereof can beformulated as a vaccine as a lipidated peptide with a covalent HTLepitope. The HTL epitope can be any peptide that has broad reactivity tohuman MHC class II to stimulate a classic helper response. Suchmolecules include but are not limited to amino acids 830-843 fromtetanus toxin (P. Panina-Bordignon et al., Eur. J. Immun. 19:2237-2242(1989)), HTL epitopes from HIV envelope protein (J. A. Berzofsky et al.,J. Clin: Invest. 88:876-884 (1991)), or a synthetic version (PADRE)predicted from known anchor residues (J. Alexander et al., Immunity1:751-761 (1994)).

[0121] The lipidation of the HTL+CTL epitope preferably is performed onthe amino terminus of the HTL epitope, with the HTL epitope being aminoterminal to the CTL epitope. Suitable lipid moieties are known anddescribed in the literature. (H. Schild et al., Eur. J. Immunol.21:2649-2654 (1991); A. Vitiello et al., J. Clin. Invest. 95:341-349(1995); K. Deres et al., Nature 342:561-564 (1989)). Alternatively, theCTL epitope can be lipidated at its amino terminus, followed by the HTLepitope, or the lipid can be found at the carboxyl terminus followed byeither the CTL or HTL epitope(s). A three amino acid spacer or any otherspacer can be inserted between the HTL and CTL epitope, or the epitopescan be fused directly in frame. Alternatively the CTL epitope lipidatedon its amino terminus can be administered together with the HTL epitope,without covalent attachment. The vaccine epitopes, regardless of primarystructure, may be injected s.c. into the forearm in a standardformulation buffer (e.g., PBS or PBS/10% DMSO or higherconcentration/0.01% triflouroacetic acid or other acid or alcohol of thesame or different concentration) once. Three to six weeks later, abooster injection of the same material may be administered. Multiplebooster injections spaced three to six weeks apart can be subsequentlyadministered, if necessary.

[0122] This injection series can be administered to a patient or at-riskindividual, or to the donor of a bone marrow transplant, who is eitherpositive or negative for the virus. Illustrative examples of lipidatedvaccine peptides include, for example: TABLE 4-------------------------------------------------- N-terminal C-terminal(Pam)₂-KSSQYIKANSKFIGITE AAANLVPMVATV (SEQ ID NO: 33)(Pam)₃-CSSQYIKANSKFIGITE AAANLVPMVATV (SEQ ID NO: 34)(Pam)₂-KSSAKXVAAWTLKAAA GGGNLVPMVATV (SEQ ID NO:35)(Pam)₃-CSSAKXVAAWTLKAAA GGGNLVPMVATV (SEQ ID NO:36)-------------------------------------------------

[0123] wherein X is cyclohexylalanine or phenylalanine. As is the casethroughout, all amino acids are represented by their universalone-letter code. “Pam” is palmitic acid. The three-A or -G spacer(underlined) can be interchanged among vaccine peptides. The format ofthe peptides shown above can be described (from the amino terminus) as:lipid-K/CSS—HTL epitope (italics)—amino acid spacer (underlined)—CTLepitope. The positions of the CTL and HTL epitopes may be interchanged.The CTL epitope (or a functional sequence variant thereof) may befurther modified by adding a leader sequence and/or the amino acids KDELcan be appended to the carboxyl terminus to enhance retention andtargeting into the endoplasmic reticulum. Palmitic acid or any suitablelipid may be used, including but not limited to stearic acid, myristicacid, lauric acid, capric acid and decanoic acid.

EXAMPLE 12

[0124] Use of Combined HTL and CTL Epitope, Lipidated at the AminoTerminus, as a Single Component Vaccine in Transgenic Mouse Studies

[0125] Two further molecules, each containing lipid, HTL and CTLepitopes, were constructed and tested in mice: TABLE 5------------------------------------------------------------ N-terminalC-terminal A. (Pam)₂-KSSAKXVAAWTLKAAANLVPMVATV (SEQ ID NO:37) B.(Pam)₂-KSSISQAVHAAHAEINE AAANLVPMVATV (SEQ ID NO:38)------------------------------------------------------------

[0126] Nomenclature for Table 5 can be found in the legend to Table 4.

[0127] Molecule A (SEQ ID NO: 37) is a vaccine that has the capabilityof working in mice and humans, whereas molecule B (SEQ ID NO: 38) shouldonly be functional in mice. One hundred nanomoles of each of thesevaccine peptides in 25% dimethysulfoxide in phosphate buffered salinewith 2.5% hexafluoroisopropanol were injected s.c. into separatetransgenic HLA A2.1 mice.

[0128] The methods referred to in Examples 9 and 10 were used to derivespleen cell populations containing cells which were specific againstpp65 and HCMV. The spleen cells from immunized mice were tested forrecognition and cytolysis against specific and non-specificpeptide-sensitized targets, HCMV infected and non-infected HLA A*0201and HLA-mismatched fibroblast targets. The 495 pp65₄₉₅₋₅₀₃ peptidesensitized targets were effectively lysed, whereas targets sensitizedwith a peptide derived from another protein (human p53) which binds toHLA A*0201 (P53₁₄₉₋₁₅₇) were ineffective. In addition, and importantly,HCMV-infected HLA A*0201 fibroblasts were effectively lysed, butuninfected fibroblasts or those which are HLA-mismatched were notrecognized or lysed, regardless of HCMV infection. These results wereobtained with both molecules A and B, although molecule A was the morepotent of the two. However, a second immunization (booster) of miceimmunized with Vaccine A at lesser amounts of 50 nmole resulted in adetectable immune response when a single immunization was not effective(Table 5). This stimulative effect of the booster is in agreement withB. Livingston et al., J. Immunol. 159:1383-1392 (1997)). These datashowed that the single molecule form of the vaccine functions tostimulate HCMV immunity in an animal model without any added moleculesor adjuvant other than the vaccine molecules A or B (Table 4) in liquidvehicle (e.g., PBS/DMSO/HFIP). The data also demonstrated that thevaccine is capable of stimulating a de novo immune response to HCMV in aHCMV-naive mouse.

EXAMPLE 13

[0129] Additional HCMV pp65 CTL Epitopes Specific for HLA A and BAlleles

[0130] There are over 100 known HLA Class I alleles of the A and Bgenes. (J. G. Bodmer et al., Tissue Antigens 49:297-321 (1997)). Using acombination of predictive algorithms and truncation analysis, additionalpeptides from pp65 that sensitize both autologous and allogeneic cellsto be lysed by MHC-restricted human CTL were identified. As discussed inExample 1, the CTL arise from HCMV seropositive humans; therefore, thedefined epitopes from pp65 were those used by humans to suppressendogenous HCMV reactivation or viremia. To define CTL reactive againstHCMV pp65 in combination with specific HLA alleles, individual clonedCTL lines were tested for recognition of EBVLCL infected with pp65vacwhich contained one of the HLA A or B alleles found in the individualwhose blood was used to derive the CTL. In cases where at least one HLAallele is shared between the EBVLCL targets and the CTL, sensitizationfor recognition and lysis was observed. This experiment was repeatedwith at least three independent cell lines containing the restrictingallele. Table 6 shows the pp65 epitopes, their HLA restriction, thenumber of independent cell lines of the same restriction which weresensitized, and the method(s) of delineation of the epitopes. TABLE 6HLA A or B Number of Allele Test Cell Method(s) of Minimal CytotoxicSpecificity Lines Determination Epitope Sequence HLA A*0101 and 3 A, B,C, D, E YSEHPTFTSQY subtypes (SEQ ID NO:3) HLA A*6801 and 3 A, B, D, EFVFPTKDVALR subtypes (SEQ ID NO:5) HLA B7 and 5 A, B, D, E TPRVTGGGAMsubtypes (SEQ ID NO:7) HLA B*3502, 04, 3 A, B, C, D, E FPTKDVAL 06 andsubtypes (SEQ ID NO:9) HLA B7 and 5 A, B, C, E RPHERNGFTVL subtypes (SEQID NO:10) HLA A*1101 and 5 A, B, C, E SVLGPISGHVLK subtypes (SEQ IDNO:11) HLA A*0201 and 5 A, B, C, D, E NLVPMVATV subtypes (SEQ ID NO:1)HLA B*3801/2 3 A, B, E PTFTSQYRIQGKL (SEQ ID NO:12) HLA B*44XX 2 DEFFWDANDIY (SEQ ID NO:13) HLA A*2402 4 A, B, E FTSQYRIQGKL (SEQ IDNO:14)

EXAMPLE 14

[0131] Vaccine Molecules Comprised of More than One CTL Epitope

[0132] In accordance with the procedures described herein, vaccinescontaining a combination of pp150 and/or pp65 epitopes, specific for thesame or different HLA Class I restriction elements, were prepared. Forvaccination of humans there is no necessity for each epitope to have thesame MHC restriction. A vaccine molecule which targets two or more MHCrestriction elements may be preferred because it allows the productionof fewer vaccine molecules, and still ensures that most HLA alleles weretargeted in a polymorphic population. Peptides with CTL epitopes whichare restricted by frequently expressed HLA alleles (see Tables 1 and 6)are preferred, and polypeptide vaccines containing epitopes from boththe pp65 and pp150 proteins, as well as an HTL epitope, are especiallypreferred for human vaccination against HCMV. The HLA alleles shown inTables 1 and 6 are a subset of possible HLA A, B, and C CTL epitopes tobe included in a multiple CTL epitope vaccine molecule. The inclusion ofmultiple CTL epitopes and an HTL epitope will lengthen the peptides (inthe range of 40-50 amino acids).

[0133] Alternatively, HCMV polypeptide vaccines may be prepared withouta covalently attached HTL epitope by using multiple CTL epitopes with aspacer of three alanine residues or another combination of amino acidsbetween each epitope, and two palmitic acid-lysyl amides at theN-terminus, as shown in Table 3.

[0134] The hydrophobicity of the sequence is an important factor. Toreduce hydrophobicity, lipid modification may be omitted. In addition,multiple HLA epitopes may be formulated into a cocktail vaccine.Preferably, each individual CTL epitope in the mixture would be fused toa CD4 T-cell epitope as described above. Thus, the cocktail vaccinecould be formulated to contain epitopes sufficient to cover about 75% toabout 90% of a multi-ethnic population using the sequences shown inTable 6.

EXAMPLE 15

[0135] Immunization of BMT Patients

[0136] A therapeutically active form of an antigenic peptide accordingto the present invention is administered to an HCMV-seropositive bonemarrow transplant donor at a sufficient time (six to eight weeks, forexample) in single or multiple doses separated by a given number of daysor weeks prior to bone marrow transplant to enable the development of ananti-HCMV cellular immune response. The antigenic peptide can beformulated in per se known manners (for example, as a lipidated peptide,optionally in combination with a helper peptide and/or an adjuvant) andwill be administered, preferably, in multiple doses. Peptides in whichthe helper peptide and antigenic peptide are linked together in onesequence, for example are suitable for this purpose.

[0137] An additional vaccine regimen consists of priming a donor with amodified Vaccinia Ankara (MVA) containing a polynucleotide viralvaccine, for example, full-length pp65, pp65 fragments or one or more ofthe CTL epitopes shown in Table 6, followed by boosting with a peptidevaccine as described in Examples 12 and 14. Those of skill in the artare fully able to devise schemes for vaccination using variouscombinations of peptide and virus administration, and these variationsare contemplated for use with the present invention. If an unmanipulatedBMT graft will be given to the recipient, such a graft will contain 25%or more of mature T cells. The T cells confer active immunity to the BMTrecipient patient. Alternatively, when a T-cell depleted BMT graft is tobe employed, an aliquot of T cells from the immunized donor can beadministered to the patient following (for example, approximately 21 to35 days) BMT to provide the recipient patient with HCMV immunity.

EXAMPLE 16

[0138] In Vitro Assay for HCMV Infected Cells

[0139] The peptides of the present invention are used in an in vitroassay to detect the presence or absence of HCMV-infected cells obtained,from a patient whose HCMV status (infected or uninfected) is unknown. Tlymphocytes obtained from the patient are incubated with antigenpresenting cells primed with a peptide of the present invention. Theactivation of CTL or CTLp reveals that the patient was infected withHCMV.

EXAMPLE 17

[0140] Induction of Immune Response by Vaccination of Transgenic Micewith Unlipidated Peptide

[0141] Two HLA-A2.1k^(b) transgenic mice were innoculated with 100nmoles of the A2 HCMV pp65 peptide NLVPMVATV (SEQ ID NO: 1) in 10% DMSO,0.1% acetic acid with 20 μg of the T-helper peptide, PADRE(AKXVAAWTLKAAA where X signifies cyclohexylalanine; SEQ ID NO: 29),emulsified in a 1:1 ratio with incomplete Freund's adjuvant bysonication for 15-30 seconds. To account for loss during transfer, 300μL or more excess emulsified vaccine over that required for injectionswas prepared. The peptide vaccine (100 μL total volume emulsifiedpreparation above) was injected subcutaneously at the base of the tail,50 μL on each side, avoiding the lateral tail veins. Positive controlanimals were injected in the same manner with the unrelated HLA-A2.1restricted hu P53₁₁₄₇₋₁₅₇ peptide and PADRE. A booster was administeredseven days later. One week after the booster immunization, splenocyteswere harvested, cultured, and stimulated in vitro. For cultivation, thecells were isolated from the spleens and suspended in 40 mL medium andcounted. Erythrocytes were omitted from the counting. The cells werethen brought to a concentration of three million cells per milliliter inmedium containing 20% Rat-Stim (Collaborative Biomedical Cat #40115,Waltham, Mass.), and plated at 1 mL/well in 24-well plates. To stimulatethe cells, 1 mL suspended antigen-presenting cells (at a ratio of 3immunized splenocytes to 1 antigen presenting cell) were added to theculture. The cells were incubated at 37° C. for seven days. Fresh mediumwas added as necessary. Stimulations were performed every seven daysafter plating by addition of 1 mL suspended antigen presenting cells asabove.

[0142] Antigen presenting cells were prepared as follows. For eachimmunized group of mice, the spleens of three mice were removedaseptically and suspended at a concentration of 1.0-1.5×10⁶ cells per mLin RPMI with 10% FCS containing 25 μg/mL LPS and 7 μg/mL dextran sulfatefor three days at 37° C. in 5% CO₂. The cells then were collected,sedimented, resuspended in serum-free medium and counted. Cells weredispensed in volumes containing 25 million cells into 15 mL conicalcentrifuge tubes and gently sedimented at 1200 rpm for six minutes. Thesupernatant was aspirated, leaving 100 μL in the tube. Peptide (SEQ IDNO: 1) (100 μM) was added to the tube, mixed well and incubated fourhours at 37° C. and 5% CO₂. The cells were resuspended in 2 mL completemedium (RPMI 1640 containing 10-12 mM HEPES, 2 mM L-glutamine, 100 U/mLpenicillin, 100 U/mL streptomycin, 50 μM β-mercaptoethanol and 10%heat-inactivated fetal calf serum) per tube and concentrated in sterilePBS. The cells were then irradiated with 2500 rads from a ¹³⁷Cs sourceand then brought to a concentration of 1 million cells per mL incomplete medium for use in stimulating the immunized splenocytes.

[0143] Six to seven days after in vitro stimulation, immunizedsplenocytes (effector cells) were collected and the cytotoxic activityof the cells was assayed against peptide-loaded or untreated targetcells using a ⁵¹Cr release assay. A bulk cell line specific forpp65₄₉₅₋₅₀₃ (SEQ ID NO: 1) derived from HLA-A2.1k^(b) immunized mice andstimulated weekly with the peptide was used as a positive control line.T2 cells were used as targets, either unloaded or loaded with either thepeptide of SEQ ID NO: 1 or a human control peptide derived from p53. SeeExample 9. To prepare the targets for the chromium release assay, T2cells in the log phase of growth were sedimented and resuspended in 10mL LCL medium (RPMI 1640 containing 1% HEPES, 10% fetal calf serum, 2%L-glutamine and antibiotics) for each of unloaded or loaded groups. Thecells were counted, resedimented, and suspended in 1 mL or less LCLmedium. Cells were either unloaded or were loaded with 10 μl of a 5 mMsolution of the appropriate peptide as described above for stimulatingantigen-presenting cells. All cells were loaded with 20 μl ⁵¹Cr stocksolution containing 10 mCi/mL Na₂ ⁵¹CrO₄. The tubes of cells then wereincubated in a 37° C. waterbath for 45-60 minutes, shaking gently every15 minutes. The cells then were washed three times in 10 mL RPMI andresuspended in 1 mL LCL medium. The target cells were recounted andsuspended in appropriate volumes of LCL medium to achieve 20,000cells/mL, and then placed in a 96-well plate at 100 μL per well.

[0144] Effector cells (immunized splenocytes) cells were brought to 2million cells per mL in RPMI containing 10% FCS and added to the wellscontaining target cells at different Effector:Target ratios as indicatedin FIG. 4. Medium alone (100 μL) was added to negative control wells tomeasure spontaneous release of ⁵¹Cr, and medium containing 2% sodiumdodecyl sulfate (100 μL) was added to positive control wells to measuretotal possible release of ⁵¹Cr. The plate was subjected to 600 rpm forsix minutes with no break in a Sorvall RT7 centrifuge (Kendro LaboratoryProducts; Newtown, Conn.) and incubated for four hours at 37° C. and 5%CO_(2,) then resedimented for six minutes at 1200 rpm. The supernatantsthen were harvested and the radioactivity measured with a Packard CobraII gamma counter (Packard Instruments; Meriden, Conn.). Specific CTLactivity was calculated as follows:${\% \quad {specific}\quad {release}} = {100 \times {\frac{\left( {{{experimental}\quad {release}} - {{spontaneous}\quad {release}}} \right)}{\left( {{{total}\quad {release}} - {{spontaneous}\quad {release}}} \right)}.}}$

[0145] Experiments in which spontaneous release was 30% or greater ofmaximum total release were discarded.

[0146]FIG. 4A shows results after one in vitro stimulation, FIG. 4Bshows results after two in vitro stimulations, and FIG. 4C shows resultsafter four in vitro stimulations. In this figure and those which follow,“T2 A2 pulsed” indicates cells presenting the peptide of SEQ ID NO: 1,“T2 p53 pulsed” indicates cells presenting the human control peptideP53₁₄₉₋₁₅₇. The data showed that the vaccination with the inventivepeptide vaccine did result in specific CTLs which could recognize andkill appropriate targets. Results improved with a second in vitrostimulation.

EXAMPLE 18

[0147] Comparison of Lipidated Vaccine Formulations

[0148] Mice were vaccinated as described in Example 17 with fourdifferent formulations of a vaccine containing(Pam)₂-KSSAKXVAAWTLKAAANLVPMVATV wherein X signifies cyclohexylalanine(SEQ ID NO: 37). Splenocytes were assayed. Effector-to-target ratios inthe chromium release assays were as indicated in FIG. 5, where theresults are shown. FIG. 5A provides results for mice innoculated with100 nmoles peptide in a solution containing 99.9% DMSO and 0.1% formicacid. FIG. 5B shows data for mice vaccinated with 100 nmoles peptidesuspended in a vehicle containing 50% DMSO, 49.9% PBS and 0.1% formicacid. FIG. 5C refers to mice injected with 83.3 nmoles peptide dissolvedin 41.7% DMSO, 57.7% PBS and 0.6% formic acid. FIG. 5D provides resultsfor mice injected with 83.3 nmoles peptide suspended in 66.7% DMSO,26.7% acetic acid and 6.7% Tween 20. The 99.9% DMSO solution vaccineprovided mice splenocytes having the greatest cytoxicity versus control.

EXAMPLE 19

[0149] Vaccination of Transgenic Mice with Monolipidated VaccineContaining PADRE

[0150] Mice were vaccinated and the cells assayed as described abovewith increasing amounts of (Pam)₂-KSSAKXVAAWTLKAAANLVPMVATV (wherein Xsignifies cyclohexylalanine (SEQ ID NO: 37). The peptide was provided ina solution in 80% DMSO, 20% PBS and 0.08% formic acid in the amountsshown in FIG. 6. Vaccination with 100 or 150 nmoles peptide resulted inpotent specific cytotoxicity, with 150 nmoles providing superiorresults.

EXAMPLE 20

[0151] Specificity of Cytotoxicity

[0152] Mice were vaccinated as described above with 150 or 25 nmoles ofthe peptide of SEQ ID NO: 37 formulated as in Example 19. Splenocyteswere assayed for cytotoxicity of T2 cells pulsed with and presentingseveral different peptides. See FIG. 7. “T2 177 pulsed” indicates cellspresenting the amidated peptide of SEQ ID NO: 39 (NLVPMVATV-NH₂); and“T2 118 pulsed” indicates cells presenting the amidated peptide of SEQID NO: 40 (YLVPMVASV-NH₂). “T2 193 pulsed” indicates cells presentingthe amidated peptide of SEQ ID NO: 41 (YLVPMVATV-NH₂). The resultsshowed a high degree of specificity.

EXAMPLE 21

[0153] Comparative Data for Monolipidated Vaccine in Different DMSOFormulations

[0154] Mice were immunized and their splenocytes assayed as described inExample 17 with 100 or 150 nmoles SEQ ID NO: 37 in vehicles containingdifferent DMSO concentrations. See FIG. 8 for the results of chromiumrelease assays. FIG. 8A: 100 nmoles in 80% DMSO, 20% PBS and 0.08%formic acid; FIG. 8B: 100 nmoles in 80% DMSO, 60% PBS and 0.04% formicacid; FIG. 8C: 150 nmoles in 80% DMSO, 20% PBS and 0.08% formic acid;FIG. 8D: 150 nmoles in 40% DMSO, 60% PBS and 0.04% formic acid.Formulations of the monolipidated vaccines were more potent whenprovided in sufficient DMSO concentration to completely solubilize thevaccine peptide. Without wishing to be bound by theory, it is believedthat concentrations of DMSO which result in complete solubility of thelipidated peptide vaccines provide superior results.

EXAMPLE 22

[0155] Vaccination Experiments with Differing DMSO Concentrations

[0156] Mice were immunized and their splenocytes assayed as described inExample 17 with 100 nmoles SEQ ID NO: 37 in the following vehiclescontaining decreasing amounts of DMSO. FIG. 9A: 80% DMSO, 20% PBS and0.02% formic acid; FIG. 9B: 70% DMSO, 30% PBS and 0.07% formic acid;FIG. 9C: 60% DMSO, 40% PBS and 0.06% formic acid; FIG. 9D: 50% DMSO, 50%PBS and 0.02% formic acid. The formulation for FIG. 9A was a solutionand the remaining formulations containing less DMSO were suspensions.The results in FIG. 9D reflect mice which received a booster, whereasthe remaining panels of FIG. 9 present data from mice receiving onevaccination only. These data confirm the hypothesis that the vaccineshould be fully solubilized for best results.

EXAMPLE 23

[0157] Vaccination Experiments with Different DMSO Concentrations

[0158] The method of Example 22 was repeated using the followingformulations. FIG. 10A: 80% DMSO, 20% PBS and 0.02% formic acid; FIG.10B: 70% DMSO, 30% PBS and 0.02% formic acid; FIG. 10C: 60% DMSO, 40%PBS and 0.02% formic acid; FIG. 10D: 50% DMSO, 50% PBS and 0.02% formicacid. As before, the vaccine preparation used to produce the data inFIG. 10A was a solution while the remaining formulations weresuspensions. The mice providing the splenocytes assayed in FIG. 10Dreceived a booster immunization.

EXAMPLE 24

[0159] Effect of Booster Immunizations

[0160] Mice were immunized and their splenocytes assayed as described inExample 17 using one or two immunizations of 25 or 50 nmoles SEQ ID NO:37. See the results in FIG. 11. FIG. 11A: 50 nmoles; FIG. 11B: 25nmoles; FIG. 11C: 50 nmoles, plus one booster; FIG. 11D: 50 nmoles, plusone booster. A booster injection increased the cytotoxic effectsachieved by the immunization for the 50 nmole dose, but apparentlyprovided little or no improvement for the 25 nmole dose.

EXAMPLE 25

[0161] Effect of Variable Formic Acid Concentrations

[0162] Mice were immunized and their splenocytes were assayed asdescribed in Example 17 using one immunization of SEQ ID NO: 37 in 80%DMSO, 20% PBS, containing 0.08% formic acid (FIGS. 12A and 12B) or 0.02%formic acid (FIGS. 12C and 12D). The amount of peptide injected was 150nmoles (FIG. 12A), 100 nmoles (FIG. 12B), 50 nmoles (FIG. 12C) or 25nmoles (FIG. 12D). Larger doses clearly resulted in higher specificcytotoxicity after vaccination.

EXAMPLE 26

[0163] Vaccination Using Unlipidated Peptide Linked to PADRE

[0164] Mice were immunized and their splenocytes assayed as described inExample 17 using one injection of the HCMV peptide of SEQ ID NO: 1,directly linked to PADRE to form one sequence (100 nmol;KSSAKXVAAWTLKAAANLVPMVATV wherein X is cyclohexylalanine; SEQ ID NO: 37)emulsified with incomplete Freund's adjuvant. The vaccine administeredwas 100 μL of an emulsion containing 5% DMSO, 45% normal saline and 50%incomplete Freund's adjuvant. Tested using T2 cells presenting thepeptide of SEQ ID NO: 1 stimulated twice in the presence of 10%Rat-Stim, the immunized splenocytes exhibited a specific cytotoxicity of11.22% at an E/T ratio of 100 and 3.18% at an E/T ratio of 20. Theimmunized splenocytes exhibited an average % cytotoxicity of −4.44% and−0.10% for T2 cells presenting the control p53 peptide, 3.31% and 3.44%for T2 cells presenting the peptide of SEQ ID NO: 39, and −6.39% and−1.81% for untreated T2 cells.

EXAMPLE 27

[0165] Vaccination Using Unlipidated Linked Peptide in a DNA Adjuvant

[0166] Two mice were immunized once with 100 nmoles the peptide of SEQID NO: 37 with 50 μg DNA adjuvant containing CpG sequences (positive DNAadjuvant). The DNA sequence used was (1826) 5′ TCCATGACGTTCCTGACGTT 3′(SEQ ID NO: 42), as described in Z. Moldoveanu, Vaccine16(11/12):1216-1224 (1998). The chromium release assay data from theseimmunized splenocytes are provided below in Table 7.

EXAMPLE 28

[0167] Vaccination Using Unlipidated Linked Peptide in a DNA Adjuvant

[0168] Example 27 was repeated using a DNA adjuvant which lacked CpGsequences (negative DNA adjuvant). The DNA sequence used was (1982) 5′TCCAGGACTTCTCTCAGGTT 3′ (SEQ ID NO: 43), as described in Z. Moldoveanu,Vaccine 16(11/12):1216-1224 (1998). The chromium release assay data arepresented below in Table 7. TABLE 7 Cytotoxicity of Cells Immunized withUnlipidated Linked Peptide Vaccine in DNA Adjuvant % CytotoxicityPresented Peptide E/T = 100 E/T = 20 Positive DNA Adjuvant A2 54.72%27.79% 177 13.23% 3.32% p53 −4.87% −2.61% untreated −6.24% 2.77%Negative DNA Adjuvant A2 45.57% 18.34% 177 22.25% 9.55% p53 −3.46%−5.82% untreated 1.52% 2.64%

[0169] Although certain preferred embodiments and examples of theinvention have been described, the invention is not so limited. Personsskilled in this field of science will understand that the presentinvention is capable of wide application in the fields of diagnosticsand therapeutics, and that modifications and variations can be made tothe invention without departing from its spirit and scope.

1. A diagnostic reagent which comprises a peptide selected from thegroup consisting of SEQ ID NOS: 1-14 and 26-28 and a label.
 2. Adiagnostic reagent which comprises a peptide according to SEQ ID NO: 1and a label.
 3. A diagnostic reagent according to claim 1 whichcomprises antigen presenting cells that present a peptide selected fromthe group consisting of SEQ ID NOS: 1-14 and 26-28.
 4. A diagnosticreagent according to claim 2 which comprises antigen presenting cellsthat present a peptide according to SEQ ID NO:
 1. 5. A method ofidentifying, in a sample of T cells, T cells that recognize HCMV, whichcomprises contacting said sample of T cells in vitro with a diagnosticreagent which comprises a peptide selected from the group consisting ofSEQ ID NOS: 1-14 and 26-28.
 6. A method according to claim 5 whereinsaid peptide is SEQ ID NO:
 1. 7. A method of identifying, in a sample ofT cells, T cells that recognize HCMV, which comprises contacting saidsample of T cells in vitro with a diagnostic reagent which comprisesantigen presenting cells that present a peptide selected from the groupconsisting of SEQ ID NOS: 1-14 and 26-28.
 8. A method according to claim7 wherein said diagnostic reagent comprises antigen presenting cellsthat present a peptide according to SEQ ID NO:
 1. 9. A method ofdiagnosing exposure to HCMV in a patient which comprises: (a) obtaininga sample of T cells from said patient; (b) contacting said sample of Tcells in vitro with a diagnostic reagent which comprises a peptideselected from the group consisting of SEQ ID NOS: 1-14 and 26-28; and(c) determining whether said diagnostic reagent binds to T cells in saidsample of T cells.
 10. A method according to claim 9 wherein saiddiagnostic reagent comprises a peptide according to SEQ ID NO:
 1. 11. Amethod of diagnosing exposure to HCMV in a patient which comprises: (a)obtaining a sample of T cells from said patient; (b) contacting saidsample of T cells in vitro with a diagnostic reagent which comprisesantigen presenting cells that present a peptide selected from the groupconsisting of SEQ ID NOS: 1-14 and 26-28; and (c) determining the degreeto which said diagnostic reagent stimulates T cells in said sample of Tcells.
 12. A method according to claim 11 wherein said peptide is SEQ IDNO:
 1. 13. A method of determining the degree of HCMV immunostimulationin a patient suffering from HCMV infection which comprises: (a)obtaining a sample of T cells from said patient; (b) contacting saidsample of T cells in vitro with a diagnostic reagent which comprisesantigen presenting cells that present a peptide selected from the groupconsisting of SEQ ID NOS: 1-14 and 26-28; and (c) determining the degreeto which said diagnostic reagent stimulates T cells in said sample of Tcells.
 14. A method according to claim 13 wherein said peptide is SEQ IDNO: 1.